Linköping University Post Print

Regulation of apoptosis-associated lysosomal

membrane permeabilization

Ann-Charlotte Johansson, Hanna Appelqvist, Cathrine Nilsson, Katarina Kågedal,

Karin Roberg and Karin Öllinger

N.B.: When citing this work, cite the original article.

The original publication is available at www.springerlink.com:

Ann-Charlotte Johansson, Hanna Appelqvist, Cathrine Nilsson, Katarina Kågedal, Karin

Roberg and Karin Öllinger, Regulation of apoptosis-associated lysosomal membrane

permeabilization, 2010, APOPTOSIS, (15), 5, 527-540.

http://dx.doi.org/10.1007/s10495-009-0452-5

Copyright: Springer Science Business Media

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Postprint available at: Linköping University Electronic Press

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-55061

REGULATION OF APOPTOSIS-ASSOCIATED LYSOSOMAL

MEMBRANE PERMEABILIZATION

Ann-CharlotteJohansson1, Hanna Appelqvist2, Cathrine Nilsson1,2,

Katarina Kågedal2, Karin Roberg1,3, Karin Öllinger2

1 Division of Otorhinolaryngology, Linköping University Hospital, Linköping, Sweden

2 Division of Experimental Pathology, Department of Clinical and Experimental Medicine,

Linköping University, Linköping, Sweden

3 Division of Otorhinolaryngology, Department of Clinical and Experimental Medicine,

Linköping University, Linköping, Sweden

Correspondence: Ann-Charlotte Johansson, Division of Otorhinolaryngology, Linköping

University Hospital, SE-581 85 Linköping, Sweden

Phone: +46-13-221525, Fax: +46-13-221529, E-mail: ann-charlotte.johansson@liu.se

ABSTRACT

Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell

death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where

they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on

an early release of cathepsins, while under other circumstances LMP occurs late in the cell

death process and contributes to amplification of the death signal. The mechanism underlying

LMP is still incompletely understood; however, a growing body of evidence suggests that

LMP may be governed by several distinct mechanisms that are likely engaged in a death

stimulus- and cell-type-dependent fashion. In this review, factors contributing to

permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal

membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described.

Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-

dependent cell death are also discussed.

KEYWORDS

Lysosome, Lysosomal release, Caspases, Calpains, Hsp, LAMP

ABBREVIATIONS

Ahr, aryl hydrocarbon receptor; ANT, adenine nucleotide translocator; apoE, apolipoprotein

E; BH3, Bcl-2 homology 3; Hsp, heat shock protein; JNK, c-Jun N-terminal kinase; LAMP,

lysosome-associated membrane protein; LAPF, lysosome-associated apoptosis-inducing

protein containing the pleckstrin homology and FYVE domains; LEDGF, lens epithelium-

derived growth factor; LMP, lysosomal membrane permeabilization; PI3K,

phosphatidylinositol-3-kinase; PLA2, phospholipase A2; ROS, reactive oxygen species;

siRNA, small-interfering RNA; SMase, sphingomyelinase; TNF, tumor necrosis factor;

TRAIL, tumor necrosis factor-related apoptosis inducing ligand; UV, ultraviolet

INTRODUCTION

Over the last decade, the lysosome has emerged as a significant component of the cellular

death machinery. Lysosomes, which were first described by de Duve and colleagues in 1955

[1], are acidic, single-membrane bound organelles that are present in all eukaryotic cells [2].

The primary function of lysosomes is degradation of macromolecules, and for this purpose,

lysosomes are filled with more than 50 acid hydrolases, including phosphatases, nucleases,

glycosidases, proteases, peptidases, sulphatases, and lipases [2]. Of the lysosomal hydrolases,

the cathepsin family of proteases is the best characterized. The cathepsins are subdivided,

according to their active site amino acids, into cysteine (cathepsins B, C, F, H, K, L, O, S, V,

W, and X), serine (cathepsins A and G), and aspartic cathepsins (cathepsins D and E) [3].

Cell death through apoptosis is tightly controlled by changes in protein expression, protein-

protein interactions, and various post-translational modifications, including proteolytic

cleavage and phosphorylation. For some of these events to occur, proteins must re-localize

from one sub-cellular compartment to another to gain access to their substrates or interacting

partners. Thus, compartmentalization is yet another important regulatory mechanism,

preventing accidental triggering of cell death signaling. The most obvious example is the

release of apoptogenic factors from mitochondria, a key event in the execution of cell death,

which is regulated mainly by proteins of the Bcl-2 family. Lysosomal hydrolytic enzymes

were initially thought to inevitably trigger necrotic cell death when released into the cytosol

[4]. However, in 1998, we discovered that the aspartic protease cathepsin D was redistributed

from lysosomes to the cytosol upon oxidative stress-induced apoptotic cell death [5] (Figure

1). Cathepsin D was the first identified lysosomal protein with apoptogenic properties, but the

subcellular location of the protein in dying cells was not addressed in the early publications

[6, 7]. In a series of papers from Brunk and co-workers, it was established that the extent of

lysosomal damage determines the cell fate; a limited lysosomal release results in cell death by

apoptosis, while massive lysosomal break-down leads to necrosis [8-12].

It is now generally accepted that apoptosis is often associated with release of cathepsins into

the cytosol, and that, in addition to cathepsin D, cysteine cathepsins B and L are involved in

apoptosis signaling [13-17]. Moreover, lysosomal destabilization, which will subsequently be

referred to as lysosomal membrane permeabilization (LMP), may also occur during other

types of cell death, including necroptosis and autophagic cell death [18, 19]. LMP can be

triggered by a wide range of apoptotic stimuli, including death receptor activation,

Figure 1. Apoptosis-associated release of lysosomal cathepsin D into the cytosol

Visualization of cathepsin D in rat cardiomyocytes exposed to the redox-cycling drug

naphthazarin by immunofluorescence microscopy (A and B) or immuno-electron microscopy

using antibodies tagged with ultra-small gold particles and subsequent silver enhancement (C

and D). There is a shift from a punctate lysosomal staining pattern in control cells (A) to a

diffuse cytosolic staining in cells exposed to naphthazarin (B). Likewise, in electron

micrographs (C), cathepsin D can be seen in lysosome-like structures in control cells (arrows),

while in cells treated with naphthazarin (D), cathepsin D is spread throughout the cytosol

(arrow heads), bars = 30 µm (A and B) and 1 µm (C and D).

endoplasmic reticulum stress, proteasome inhibition, oxidative stress, DNA damage, osmotic

stress, and growth factor starvation [13-17]. In some experimental settings, lysosomal release

is an early event essential for apoptosis signaling to proceed, while under other circumstances

LMP occurs late in the cell death process and contributes to amplification of the death signal

(Figure 2). Cathepsin relocalization seems to be critical for apoptogenicity, and it has been

demonstrated that microinjection of cathepsins into the cytosol is sufficient to trigger

apoptotic cell death [20-22]. Cathepsin release may result in caspase-dependent or -

independent cell death with or without involvement of mitochondria [13, 17]. Caspases-2 and

-8, phospholipase A2 (PLA2), sphingosine kinase-1, and the Bcl-2 family proteins Bid, Bcl-2,

Bcl-XL, and Mcl-1 may all be downstream targets of cathepsins. A detailed description of the

mechanisms by which lysosomal cathepsins propagate the apoptotic signal can be found

elsewhere [13-16].

Figure 2. Lysosomal participation in cell death signaling

Lysosomal membrane permeabilization (LMP), i.e., release of cathepsins from lysosomes into

the cytosol, is an important step in cell death signaling induced by a variety of stimuli, such as

death receptor activation, radiation, and cytotoxic drugs. Several cytosolic cathepsin targets

have been described and the signaling downstream LMP involves both caspase-dependent and

-independent pathways. Engagement of the mitochondrial pathway is a common downstream

event of LMP, but cathepsins may also cause cell death without involvement of mitochondria.

It is still unclear whether a subset of lysosomes is particularly susceptible to apoptosis-

associated LMP and thus release all of their contents while other lysosomes release none, or if

all lysosomes within a given cell are equally affected but release only part of their contents.

There are, however, indications that interlysosomal differences regarding vulnerability to

LMP do exist [23, 24]. For example, large lysosomes appears to be more susceptible to LMP

than small ones [24].

The mechanism underlying LMP is still incompletely understood. Apart from cathepsins,

other hydrolases [25-28], H+ (causing acidification of the cytosol) [29], Ca

2+ [30], and several

of the dyes that accumulate in lysosomes with intact membranes, e.g., acridine orange [8],

Lucifer Yellow [10], and LysoTracker [31], can escape into the cytosol at the onset of

apoptosis. This may suggests a nonspecific release mechanism, such as pore formation or

limited membrane damage, rather than selective transport across the membrane, which would

likely be specific for a single group of molecules. However, it should be noted that the release

of H+, Ca

2+, and lysosomotropic dyes does not necessarily reflect a change in membrane

integrity, but could merely be due to a change in ion pump activity. It has been suggested that

apoptosis-associated lysosomal release is size-selective, as 10- and 40-kDa FITC-dextran

molecules are released from lysosomes during staurosporine-induced T-cell apoptosis, while

70- and 250-kDa FITC-dextran molecules are retained [32]. Nevertheless, that upper limit of

size-selection does not apply in all death models, as release of the 150 kDa lysosomal protein

N-acetyl-β-glucosaminidase has been observed under other experimental conditions [25-27].

Conceivably, LMP may be governed by several distinct mechanisms, each with its own

characteristics, which are likely engaged in a death stimulus- and cell-type-dependent fashion.

The mechanisms that have been suggested to contribute to permeabilization of the lysosomal

membrane are described in the following sections, as are those that are thought to safeguard

lysosomal integrity and confer resistance to lysosome-dependent cell death.

PROMOTERS OF LYSOSOMAL MEMBRANE PERMEABILIZATION

Viral and bacterial proteins

Apoptosis triggered by bacterial and viral infections sometimes involves LMP, and in certain

contexts the participation of cathepsins is required for apoptotic signaling to proceed [33-38].

The human immunodeficiency virus type 1 (HIV-1) protein Nef is one of the non-mammalian

proteins that, when entering mammalian cells, causes permeabilization of the lysosomal

membrane [36]. Overexpression of Nef elicits an apoptotic response in CD4+

T lymphocytes

via disruption of lysosomal integrity, thus, the progressive and massive destruction of CD4+

T

lymphocytes characteristic of HIV-1-related disease may in part be due to Nef-induced LMP.

Parvovirus H1 is another virus that induces cell death characterized by translocation of

cathepsins to the cytosol [34]. A small amount of viral proteins were found within the

lysosomal fraction, and it was speculated that the capsid protein VP1, which possesses a

domain displaying phospholipase A2 (PLA2) activity, could be involved in induction of LMP.

The diphtheria toxin is one of the most interesting examples of a bacterial protein that can

permeabilize the membranes of the endolysosomal compartment. After entering the cell via

endocytosis, one of the subdomains of the toxin inserts into the endosomal membrane and

forms pores through which the catalytic subunit of the toxin passes into the cytosol [39-42]. In

the same way, components of the tetanus [43], botulinum [44], and anthrax [45] toxins are

believed to permeabilize the endosomal membrane to gain access to the cytosol. As discussed

below, certain mammalian proteins involved in apoptosis signaling share high sequence

homology with these viral proteins, and may well be key regulators of apoptosis-associated

LMP.

Pro-apoptotic Bcl-2 family proteins

The Bcl-2 family consists of both pro- (e.g., Bax, Bak, Bid, Bad, Bim, Noxa, and Puma) and

anti-apoptotic (e.g., Bcl-2, Bcl-XL, and Mcl-1) proteins that are critical regulators of the

mitochondrial pathway to cell death. The multi-domain proteins Bax and Bak are pore-

forming proteins that enable the release of apoptogenic factors such as cytochrome c from

mitochondria, while the so-called BH3-only-domain proteins (e.g., Bid, Bad, Bim, Noxa, and

Puma) serve to promote activation of these multi-domain family members upon an apoptotic

insult. The main function of the anti-apoptotic Bcl-2 family proteins, on the other hand, is to

prevent the release of apoptogenic factors. Interestingly, the hypothesis that Bcl-2 family

proteins can release cytochrome c through pore formation was based on the discovery that

some of these proteins showed high structural similarity to the pore-forming domain of the

diphtheria toxin, which, as mentioned above, is known to form pores in the endosomal

membrane [46, 47]. Several years later, we discovered that Bax could also permeabilize the

lysosomal membrane, and thereby promote cell death by the release of lysosomal cathepsins

to the cytosol [26]. Confocal and immuno-electron microscopy showed that Bax translocated

from the cytosol to the lysosomal membrane upon staurosporine treatment of human

fibroblasts. Furthermore, recombinant Bax was inserted into the membrane of isolated rat

liver lysosomes and caused the release of lysosomal constituents. These data provided the first

evidence that Bax is mechanistically involved in LMP and are now supported by a growing

number of publications demonstrating involvement of multiple pro- and anti-apoptotic Bcl-2

family proteins in the regulation of lysosomal membrane integrity.

Bax is activated and relocated to lysosomes in hepatocytes upon treatment with free fatty

acids [48, 49], in cholangiocarcinoma cells in response to tumor necrosis factor-related

apoptosis inducing ligand (TRAIL) [50], and in ovarian cancer cells treated with etoposide

[51]. In all three cases, small interfering RNA- (siRNA)-mediated downregulation of Bax

prevented LMP, indicating a mechanistic importance of Bax’s lysosomal location.

Furthermore, siRNA-mediated downregulation of Bim was found to prevent TRAIL-induced

LMP [50]. Bim was activated upon TRAIL treatment and colocalized with Bax in lysosomes,

suggesting that it may trigger Bax-mediated LMP. In another study, Bid, the most well-known

Bax activator, was found to be involved in tumor necrosis factor (TNF)-α-induced lysosomal

destabilization in hepatocytes [52]. Upon TNF-α treatment, Bid was detected in lysosomes,

and cathepsin B was released to the cytosol. However, no such release was seen in Bid-/-

cells.

Even though the involvement of Bax was not considered in this study, it is tempting to

speculate that LMP was caused by Bid-mediated activation of Bax. In a recent report, BH3

domain peptides were found to permeabilize isolated lysosomes in the presence of Bax [50].

Interestingly, Bak, the other main pore-forming Bcl-2 family protein, also localizes to

lysosomes [48], but there are as yet no reports of Bak-induced LMP. Conceivably, triggering

of LMP could be a general function of pro-apoptotic Bcl-2 proteins. Similar to mitochondrial

membrane permeabilization [53], LMP-associated Bax activation seems to rely on different

BH3-only proteins depending on the death stimulus and cell type. In addition to a direct effect

of Bcl-2 proteins at the lysosomal membrane, Bcl-2 family members may regulate LMP at the

mitochondrial level as engagement of mitochondria can lead to increased lysosomal release

via positive feed-back. However, as release of cathepsins has been observed in Bax/Bak

double knockout cells there are clearly additional mechanisms independent of Bax and Bak

that contribute to LMP [54].

p53

Permeabilization of the lysosomal membrane occurs in p53-induced apoptosis [55], during

which p53 itself localizes to lysosomes and triggers LMP in a transcription-independent

manner [56]. It was proposed that p53 phosphorylated at Ser15 (p-p53Ser15

) may be recruited

to the lysosomal membrane by a newly discovered protein called Lysosome-associated

apoptosis-inducing protein containing the pleckstrin homology and FYVE domains (LAPF).

This protein, which associates with lysosomes upon induction of apoptosis, is essential for

cell death induced by TNF-α, ionizing irradiation, and the DNA-damaging drugs 5-

fluorouracil and oxaliplatin [56, 57]. Downregulation of either p53 or LAPF prevented TNF-

α-induced LMP [56]. Interestingly, silencing of LAPF expression abrogated lysosomal

translocation of p-p53Ser15

, whereas silencing of p53 expression had no effect on lysosomal

translocation of LAPF, indicating that LAPF may trigger LMP by acting as an adaptor protein

for p-p53Ser15

. Furthermore, p-p53Ser15

localizes to lysosomes in cultured cortical neurons after

exposure to the psychoactive ingredient of marijuana, ∆9-tetrahydrocannabiol [58], or β-

amyloid [59], both of which cause neuronal cell death. Also in these models of apoptosis,

LMP was abrogated by siRNA-mediated downregulation or chemical inhibition of p53. It is

worth noting that p53, under certain circumstances, localizes to the mitochondrial membrane

during apoptosis and can trigger mitochondrial membrane permeabilization by direct

interaction with Bcl-2 family members [60, 61]. It is tempting to speculate that a similar

mechanism governs p53-induced LMP. Additionally, p53 promotes Bax-mediated

mitochondrial membrane permeabilization through transcriptional upregulation of the BH3-

only-domain proteins Puma and Noxa [62-64]. In this way, p53 may engage the lysosomal

pathway without translocating to lysosomes. Recent data from our lab supports this notion;

ultraviolet B- (UVB)-induced, p53-dependent LMP is associated with an increase in Puma

and Noxa expression without the appearance of p53 at the lysosomal membrane [65].

Proteases

CASPASES

There are a number of publications suggesting that caspases, under certain circumstances, are

involved in the release of apoptogenic factors from lysosomes [50, 52, 66-69]. Caspase-8, in

particular, plays a significant role in the engagement of the lysosomal death pathway

following death receptor activation [50, 52, 67-69]. Caspase-8 can trigger the release of

cathepsins from purified lysosomes, and the effect was enhanced by the addition of a

cytosolic fraction [67], indicating that caspase-8-induced LMP is at least partly mediated by

activation of one or several cytosolic substrates. Bid, which presumably would trigger Bax-

mediated LMP, has been presented as a possible candidate [52]. In our hands, neither caspase-

8 nor activated Bid alone permeabilized the membrane of isolated lysosomes [26], suggesting

that additional factors, such as Bax or Bak, are indeed required. In another intriguing study,

caspase-8 cleaved and activated caspase-9 in an apoptosome-independent fashion [68]. By

using genetically modified mouse embryonic fibroblasts, caspase-9 was proven to be

indispensable for triggering LMP, but the exact mode of action remains elusive. Interestingly,

depletion of Bid did not prevent the loss of lysosomal membrane integrity. These data suggest

that there are at least two different mechanisms by which caspase-8 is able to induce LMP.

In a recent study, caspase-2 activation was shown to be required for LMP during endoplasmic

reticulum stress-induced apoptosis [70]. The effect was not dependent on Bax, ceramide,

Ca2+

, or reactive oxygen species (ROS) production, suggesting a more direct role of caspase-

2. Indeed, caspase-2 has previously been shown to induce cathepsin release from purified

lysosomes [67].

CATHEPSINS

It has been proposed that lysosomal proteases, in particular cathepsin B, may contribute to

their own release [31, 48, 71]. In mouse hepatocytes, lysosomal destabilization evoked by

TNF-α and sphingosine is dependent on cathepsin B, as induction of LMP fails in cathepsin

B-deficient cells [31]. Moreover, chemical inhibition of cathepsin B significantly reduces both

free fatty acid- and TRAIL-induced LMP [48, 71]. However, it is not clear whether intra- or

extralysosomal cathepsin B is involved. As cathepsin B is constitutively active inside

lysosomes, it cannot be sufficient for LMP; however, it may be necessary. Data in favor of

intralysosomal cathepsin B as a mediator of LMP was provided by cell-free experiments in

which sphingosine was found to cause lysosomal release from cathepsin B-containing but not

from cathepsin B-deficient lysosomes [31]. Alternatively, cathepsin B-mediated LMP may

constitute an amplifying feedback loop, in which a small amount of released cathepsin B

triggers more extensive LMP from outside the lysosome. The latter is supported by the

finding that upregulation of serine protease inhibitor 2A, a potent inhibitor of cathepsin B

located in the cytosol and nucleus, reduces lysosomal breakdown [72].

CALPAINS

Calpains catalyze lysosomal membrane destabilization during neuronal cell death [73-76].

Cell death induced by ischaemia or hypochloric acid is associated with an increase in

cytosolic [Ca2+

], activation of calpains, and LMP. Activated µ-calpain localizes to the

lysosomal membrane prior to release of cathepsins, indicating involvement in the triggering of

LMP [74, 75, 77]. In further support of this hypothesis, -calpain can permeabilize the

membrane of isolated lysosomes [67], and pharmacological inhibition of calpains abrogates

LMP [73]. Interestingly, calpain activity correlates with the appearance of p-p53Ser15

at the

lysosomal membrane, suggesting that calpains may, under certain circumstances, promote

p53-mediated lysosomal release [59].

Reactive oxygen species

An increased production of ROS may lead to destabilization of the lysosomal membrane via

massive peroxidation of membrane lipids. Photosensitizers located in lysosomes that generate

singlet oxygen upon radiation have been shown to cause rapid release of cathepsins to the

cytosol [78-80]. Moreover, apoptosis induced by UVA radiation or H2O2, both of which

trigger cell death mainly by generating oxidative stress, involves LMP [12, 65, 81].

Additionally, other apoptosis inducers, e.g., N-(4-hydroxyphenyl)retinamide [82] and

vacuolar ATPase inhibitors [83], cause ROS-dependent LMP. However, in the latter model

systems it is not clear whether leakage over the lysosomal membrane is due to ROS-mediated

membrane damage. Alternatively, alterations in the intracellular redox balance may elicit a

redox-dependent signaling cascade resulting in LMP.

Induction of LMP by lysosomal membrane peroxidation reactions has been extensively

studied in model systems in which the intralysosomal iron content has been manipulated [84-

88]. It is hypothesized that intralysosomal iron, originating from degraded metallo proteins,

reacts with H2O2 and generates highly reactive hydroxyl radicals that initiate peroxidation of

the lysosomal membrane (Figure 3). In an illustrative experiment, macrophages that were

allowed to phagocytose silica particles with high amounts of surface-bound iron suffered from

extensive lysosomal damage, while those that ingested silica particles pretreated with the

pharmacologic iron chelator desferrioxamine displayed only modest lysosomal leakage [89].

In several additional studies it has been established that phagocytosis of iron complexes or

iron-containing proteins increases lysosomal vulnerability, while a reduction in

intralysosomal reactive iron reduces lysosomal leakage and cell death [84-88].

Kinases

Cellular signal transduction involves a wide range of small GTPases and kinases constituting

a complex signaling network that ultimately regulates virtually all cellular events including

cell proliferation, differentiation, and cell death. Ras signaling pathways participate in the

overall regulation of lysosomal function, and phosphatidylinositol-3-kinase (PI3K), one of the

downstream targets of Ras, is indispensable for vesicular transport to lysosomes [90-92]. H-

ras driven transformation of NIH3T3 cells causes increased expression of cysteine cathepsins

and reduced lysosomal stability during TNF-induced cell death [93]. Inhibition of PI3K

sensitizes vascular endothelial cells to cytokine-induced apoptosis by increasing lysosomal

permeability [94]. Furthermore, activation of c-Jun N-terminal kinase (JNK) promotes

lysosomal breakdown in response to TNF-α, TRAIL, and UVB radiation, possibly via

activation of the BH3-only-domain protein Bim [50, 95, 96]. Additionally, in neurons

exposed to β-amyloid, phosphorylation of p53 at Ser15 is JNK1 dependent [97]. In contrast,

inhibitors of JNK fail to affect lysosomal permeability in H-ras transformed NIH3T3 cells.

Instead, activation of the Raf1-extracellular signal-regulated kinase (-ERK) pathway is

responsible for increased engagement of the lysosomal death pathway [98]. Taken together,

these data suggest that LMP is likely regulated by kinase-dependent signaling events,

although the number of studies so far is limited.

Figure 3. Destabilization of the lysosomal membrane by lipid peroxidation

In response to oxidative stress, increased amounts of hydrogen peroxide can diffuse into the

lysosome. In the lysosomal lumen, the acidic milieu and the presence of low-molecular-

weight iron, derived from degraded iron-containing proteins, promotes the reduction of iron

and the generation of hydroxyl radicals. The hydroxyl radicals induce peroxidation of

membrane lipids and thereby cause leakage of lysosomal constituents into the cytosol.

Changes in lysosomal membrane lipid composition

Obviously, the lysosomal membrane composition plays a key role in the maintenance of

lysosomal integrity. Damage to lysosomal membrane components or changes in the

membrane structure and fluidity could result in lysosomal destabilization.

PLA2, phospholipase C, and sphingomyelinase - enzymes that regulate the membrane lipid

composition - all show increased activity in the presence of high cytosolic [Ca2+

], which is

characteristic of apoptosis. Studies using isolated rat lysosomes have shown that these

enzymes [99-102] as well as the phospholipid products arachidonic acid [103],

lysophosphatidylcholine [104], and phosphatidic acid [105], affect the osmotic sensitivity of

lysosomes, making them more susceptible to osmotic stress.

PLA2 stands out as a particularly interesting candidate because it is involved in lysosomal

membrane destabilization in several different experimental systems [30, 76, 106-109]. For

example, lysosomes are permeabilized in a PLA2-dependent fashion in response to low

concentrations of H2O2 [106]. In this context, activation of PLA2 is dependent on an early

minor release of lysosomal contents (suggesting an alternative mechanism for the initial

release), and PLA2 engages in an amplifying feed-back loop. Notably, incubation of PLA2

with rat liver lysosomes results in leakage of lysosomal constituents.

Apoptosis-associated LMP may also be mediated by an increase in the sphingolipid

sphingosine [10, 31]. Ceramide is produced from sphingomyelin upon activation of

sphingomyelinases (SMase) and is further processed into sphingosine by ceramidase [110]

(Figure 4). It has been proposed that sphingosine accumulates inside lysosomes, where it

permeabilizes the membrane in a detergent-like fashion, resulting in cell death [10]. Under

certain circumstances, sphingosine-induced LMP is dependent on cathepsin B, as sphingosine

fails to permeabilize lysosomes from cathepsin B-/-

hepatocytes [31]. These data also present

the possibility that sphingosine serves as a general trigger of cathepsin B-mediated

intralysosomal events leading to LMP.

In a recent publication, proteins upregulated in lysosomes at the onset of apoptosis-associated

LMP were identified using a proteomic approach [111]. Interestingly, two of these proteins,

prosaposin and protein kinase Cδ, can lead to activation of the lysosomal enzyme acid SMase.

Protein kinase Cδ activates acid SMase after translocating from the cytosol to the lysosomal

membrane [112], and prosaposin is the precursor of four lysosomal sphingolipid activator

proteins (saposins A-D) that promote acid SMase-mediated sphingomyelin degradation [113].

Other factors

β-AMYLOID

Alzheimer’s disease is an age-related progressive neurodegenerative disease in which β-

amyloid peptides are thought to confer neurotoxicity. Accumulation of β-amyloid in

lysosomes of cultured cells results in LMP-dependent cell death [114, 115]. As β-amyloid is

amphipathic and forms micelle-like aggregates [116, 117], the toxicity of β-amyloid could be

mechanistically similar to that of lysosomotropic detergents, which self-assemble into

micelles within lysosomes [118]. Alternatively, β-amyloid, which, like some of the Bcl-2

family proteins, shows structural similarities to pore-forming bacterial toxins, may trigger

lysosomal release by creating pores. Indeed, oligomeric β-amyloid can incorporate into

artificial lipid bilayers and form pore-like structures [119, 120].

Figure 4. Accumulation of sphingosine leads to lysosomal membrane permeabilization

An apoptotic stimulus, e.g., tumor necrosis factor- , leads to increased activity of

sphingomyelinase, which catalyzes the formation of ceramide from sphingomyelin. By the

action of ceramidase, ceramide is then converted into sphingosine, which accumulates in

lysosomes and acquires detergent-like properties, leading to lysosomal membrane

permeabilization.

APOLIPOPROTEIN E

Apolipoprotein E (apoE) is involved in lipid transport and one of its three major isoforms,

apoE4, is a known risk factor for Alzheimer’s disease. ApoE4-transfected cells are more

susceptible to lysosomal leakage induced by β-amyloid as compared to untransfected cells or

cells transfected with apoE3 [121]. This effect is dependent on a low intralysosomal pH, as

overexpression of apoE4 fails to potentiate leakage from lysosomes neutralized with

bafilomycin or NH4Cl. Furthermore, at acidic pH, apoE4 binds more avidly to phospholipid

bilayer vesicles and potentiates β-amyloid-induced disruption to a greater extent. It was

suggested that, especially at a low pH, apoE4 is converted into a molten globule capable of

binding and altering membrane stability [122]. Thus, such apoE4-mediated effects on

membrane stability are likely restricted to the lysosomal membrane, and may, under certain

circumstances, play a significant role in the determination of cell fate.

ARYL HYDROCARBON RECEPTOR

In a murine hepatoma cell line in which TNF-α-induced cell death was found to be

independent of caspase-8, LMP was dependent on the aryl hydrocarbon receptor (Ahr) [123].

This receptor is a ligand-activated transcription factor whose target genes are involved in

many cellular functions, including apoptosis. In addition, it has been suggested that the Ahr

may have ligand-independent activities. Ahr deficiency prevents disruption of lysosomes

induced not only by TNF-α treatment, but also following photodynamic therapy [124].

ANT

The adenine nucleotide translocator (ANT) agonist atractyloside, which triggers opening of

the mitochondrial permeability transition pore and release of mitochondrial cytochrome c,

was shown to also induce release of cathepsin B from isolated lysosomes [125]. This was

accidentally discovered by researchers aiming to identify factors released from a

mitochondrial fraction, apparently contaminated with lysosomes. This finding suggests that

ANT-like proteins may be present in the lysosomal membrane and regulate its permeability;

however, in our hands, only a very high concentration of atractyloside permeabilized the

membrane of isolated rat liver lysosomes [26].

GRANULYSIN

The killer effector molecule granulysin, which, together with perforin and granzymes, is

located in the cytolytic granules of human cytotoxic T-lymphocytes and natural killer cells

was recently found to cause necroptosis (programmed necrosis) via LMP [19]. Granulysin,

which interacts with lipids to cause disruption of membranes, has a broad-spectrum

antimicrobial function and shows potent cytotoxic activity against tumor cells. Granulysin

enters lysosomes of tumor cells and triggers release of cathepsin B, which in turn engages the

mitochondrial pathway via cleavage of Bid. Thus, it appears that our immune system is armed

with a weapon to target lysosomes for the destruction of unwanted cells.

SAFEGUARDS OF LYSOSOMAL MEMBRANE INTEGRITY

It has been suggested that the stability of the lysosomal membrane could be modulated in

different ways, and that this could contribute to the determination of cell fate. An example

from normal physiological conditions is the selection of B-lymphocytes in the germinal

centers. B-lymphocytes with high affinity B-cell receptors are spared from apoptotic cell

death by interaction with follicular dendritic cells, which confer resistance to LMP [69]. The

underlying mechanism was not unraveled in this study, but molecules involved in the

stabilization of the lysosomal membrane have been identified by others (see below).

Furthermore, cancer cells often show various changes in lysosomal function as well as altered

susceptibility to lysosome-mediated cell death. For example, activation of the Ras or Src

oncogenes is associated with changes in the lysosomal distribution, density, size,

ultrastructure, and content, all of which may influence membrane stability [24, 90, 91, 98].

Heat shock proteins

Heat shock proteins (Hsps) are molecular chaperones essential for cells’ ability to cope with

environmental stress [126]. Interestingly, Hsp70 is found at the lysosomes of many tumor

cells and stressed cells, and has been shown to prevent LMP induced by TNF-α, etoposide,

H2O2, or UVB irradiation [27, 127, 128]. Notably, Hsp70 is frequently overexpressed in

tumors, and downregulation of Hsp70 in breast, pancreatic, or colon cancer cells leads to

LMP and cathepsin-mediated cell death without any external death stimuli [27, 129],

suggesting that the tumorigenic potential of Hsp70 could, in part, be due to stabilization of

lysosomes. Hsp70 may prevent lysosomal membrane permeabilization by scavenging of

lysosomal free iron and blocking of oxidative events [128, 130]. Moreover, it is tempting to

speculate that Hsp70, which has been shown to interact with Bax and prevent its translocation

to mitochondria [131, 132], could interfere with Bax-induced LMP.

In addition, Hsp70-2 can prevent permeabilization of the lysosomal membrane [133]. In this

case, the protective effect depends on Hsp70-2-mediated upregulation of lens epithelium-

derived growth factor (LEDGF). Knockdown of LEDGF in cancer cells induces

destabilization of lysosomal membranes and cathepsin-mediated cell death, while ectopic

expression stabilizes lysosomes and prevents cancer cell death. However, as LEDGF has not

been detected in lysosomes, the protective mechanism remains obscure.

Bcl-2 proteins

Anti-apoptotic members of the Bcl-2 family are well-known regulators of mitochondrial

membrane permeabilization, which, during recent years, have gained attention as possible

modulators of lysosomal membrane stability [49, 50, 66, 134, 135]. Our finding that LMP

may be induced by Bax is supported by the fact that overexpression of Bcl-2 prevents

lysosomal destabilization [66, 134, 135]. In two of these studies, it was suggested that Bcl-2

stabilizes lysosomes by preventing activation of PLA2 [134, 135]. However, it is tempting to

speculate that the protective effect of Bcl-2 is, at least in part, due to neutralization of pro-

apoptotic members of the Bcl-2 family. The theory that anti-apoptotic Bcl-2 proteins are

safeguards of lysosomal membrane integrity is supported by data from Gregory Gores and co-

workers [49, 50]. In these studies, Mcl-1 and Bcl-XL prevented LMP induced by TRAIL and

free fatty acids, respectively. In both cases, Bax was activated and translocated from the

cytosol to lysosomes upon apoptosis induction. Furthermore, overexpression of Mcl-1 and

Bcl-XL, as well as siRNA-mediated downregulation of Bax, stabilized the lysosomal

membrane.

Antioxidants

As described previously, ROS may compromise the integrity of lysosomes via peroxidation of

membrane lipids. A variety of protective mechanisms have evolved to scavenge ROS and

protect cells from their adverse effects; these are collectively known as the cell’s antioxidant

defense. The defense mechanisms include low-molecular-weight antioxidants, for example

vitamins C and E, coenzyme Q10, and glutathione, as well as antioxidant enzymes, such as

glutathione peroxidase, catalase, and superoxide dismutase [136]. Vitamin E, a lipid soluble

antioxidant that protects from lipid peroxidation [137], can prevent lysosomal release [5, 138].

Moreover, LMP induced by N-(4-hydroxyphenyl) retinamide or inhibitors of the vacuolar

ATPase can be abrogated by increasing the amount of intracellular cysteine, which alters the

redox balance [82, 83].

Furthermore, iron-binding proteins may serve to mitigate oxidant-induced LMP, as

intralysosomal free iron can generate highly reactive hydroxyl radicals, leading to

peroxidation of membrane lipids. Indeed, a number of studies have demonstrated that iron-

binding proteins such as ferritin and metallothionein can reduce lysosomal leakage and cell

death [84, 85, 139]. As mentioned above, also Hsp70 can bind intralysosomal redox-active

iron and may, in that way, protect the lysosomal membrane from free-radical attack and

permeabilization [128, 130].

Cholesterol and sphingomyelin

Cholesterol may play an important role in the maintenance of lysosomal membrane stability.

It has long been known that addition of cholesterol to lysosomes reduces their permeability

[140]. Accordingly, a reduced lysosomal membrane cholesterol level is associated with

increased permeability to protons and potassium ions; this, in turn, causes an osmotic

imbalance and destabilization of the lysosomal membrane [141-143]. Sphingomyelin affects

the membrane fluidity by acting as a scaffold for incorporation of cholesterol [144, 145].

Exogenous sphingomyelin, as well as the sphingomyelin derivative 3-O-

methylsphingomyelin, have been shown to accumulate in the lysosomal membrane and

protect from lysosomal membrane disruption in response to TNF-α and lysosomal

photosensitizers [78]. Thus, apoptosis-associated conversion of sphingomyelin into

sphingosine (Figure 4), a promoter of LMP, appears to have a dual negative effect on

lysosomal membrane integrity.

Lysosome-associated membrane protein-1 and -2

The lysosomal membrane contains highly glycosylated proteins whose complex intralumenal

carbohydrate side chains form a coat on the inner surface of the membrane. It has been

suggested that these glycoproteins serve as a barrier against the hydrolytic activity of the

lysosomal enzymes and thereby prevent accidental release of lysosomal constituents into the

cytosol. Lysosome-associated membrane protein-1 (LAMP)-1 and -2, which constitute ~50%

of the lysosomal membrane proteins [146, 147], can modulate the sensitivity of the lysosomal

membrane to an apoptotic insult [98]. Oncogene-induced reduction in LAMP-1 and -2 can be

caused by cathepsin B overexpression and is associated with increased susceptibility to LMP

in response to photo-oxidation and the anti-cancer drug siramesine. The authors of that

finding speculate that upregulation of lamp-1 and -2 mRNA, which has been observed in

various human cancers [148, 149], might be an attempt to compensate for the deleterious

effect of the reduced half-life of LAMP-1 and -2 in cancer cells overexpressing lysosomal

cysteine cathepsins.

CONCLUSION

A growing body of evidence suggests that apoptosis is generally associated with LMP,

occurring either as a triggering early event or an amplifying late event. With the wider

acceptance of lysosomes as part of the signaling cascade leading to apoptotic cell death, the

molecular mechanisms governing LMP have received increased attention. As outlined above,

there are many proteinaceous and non-proteinaceous factors, including Bcl-2 family proteins,

ROS, caspases, cathepsins, cholesterol, and Hsps, that influence the stability of the lysosomal

membrane, and their individual importance appears to depend on the cell type and death

stimulus (Figure 5). However, the main mechanism responsible for apoptosis-associated

LMP, if any, remains to be identified. A deeper understanding of the regulation of LMP and

other apoptosis signaling events could enable the development of therapies for diseases

associated with excessive or insufficient apoptosis such as neurodegenerative disorders and

cancer, respectively.

Figure 5. Factors regulating lysosomal membrane permeabilization

This schematic presents a number of factors that may be responsible for lysosomal membrane

permeabilization (LMP). The relative importance of each mechanism likely depends on the

cell type and death stimulus. Mechanisms that are believed to safeguard lysosomal integrity

and protect from lysosome-mediated cell death are shown in red. “Changes in membrane lipid

composition” includes membrane destabilizing factors such as phospholipase A2 and

sphingosine, as well as LPM protective substances e.g., cholesterol and sphingomyelin.

Abbreviations: JNK, c-Jun N-terminal kinase; LAPF, lysosome-associated apoptosis-inducing

protein containing the pleckstrin homology and FYVE domains; Hsp, heat shock protein;

ROS, reactive oxygen species; ANT, adenine nucleotide translocator; Ahr, aryl hydrocarbon

receptor; LEDGF, lens epithelium-derived growth factor; LAMP, lysosome-associated

membrane protein.

ACKNOWLEDGEMENTS

We thank Lotta Hellström and Fredrik Jerhammar for critical reading of the manuscript.

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